1. Field of the Invention
The present invention relates generally to the field of periodontal tissue regeneration, and particularly concerns the treatment of diseases related to periodontitis and periodontal disease. The invention provides 17 kDa sheath protein and its derived peptides, which have cementum regeneration promoting activity and are involved in periodontal regeneration and cytodifferentiation of periodontal ligament cells. Also disclosed are the methods for isolating other enamel matrix proteins, and various therapeutic methods using the compositions of the invention.
The invention provides a cementum regeneration promoting protein segment comprising an isolated, synthesized and recombinant sheath protein, polypeptide, or peptide. In certain embodiments of the present invention, the sheath protein is a porcine sheath protein. In other embodiments of the present invention the sheath protein is a human, murine, rat, bovine, sheep, monkey and every mammal sheath protein.
As used herein in the context of various of the instant compositions and methods, the term “protein” will be understood to mean a proteinaceous segment that is longer than about 150 contiguous amino acids and in most aspects comprises more than about 70% of the amino acids encoded by a gene. As used herein in the context of various of the instant compositions and methods, the term “polypeptide” will be understood to mean a proteinaceous segment that is longer than about 50 contiguous amino acids in length, and the term “peptide” will be understood to mean a proteinaceous segment that is between about 3 and about 50 contiguous amino acids in length. Thus sheath protein proteinaceous segment of varying overall length that retain signaling, regulatory and structural properties and functions are provided herein.
As used herein in the context of various of the instant compositions and methods, the term “sheath protein” or the term “sheathlin” will be understood to include wild-type, polymorphic and mutant versions of sheath protein or sheathlin sequences. Wild-type sequences are defined as the first identified sequence; polymorphic sequences are defined as naturally occurring variants of the wild type sequence that have no effect on the expression or function of the sheath protein or sheathin proteins, polypeptides, or peptides or domain thereof; and mutant sequences are defined as changes in the wild-type sequence, either naturally occurring or introduced by the hand of man, that have an effect on either the expression and/or the function of the sheath protein or sheathlin proteins, polypeptides or peptides or domains thereof. The invention thus also includes the provision of DNA segments vectors, genes and coding sequence region that encode various forms of sheath protein or sheathlin proteins, polypeptides, peptides or any fusion protein, polypeptides or peptide thereof.
As used herein in the context of various of the instant compositions and methods, the term “cementum regeneration promoting protein” will be understood to include the proteins, polypeptides and peptides containing amino acid sequences shown a cementum regeneration promoting activity in sheath protein sequences. Wild-type sequences are defined as the first identified sequence; polymorphic sequences are defined as naturally occurring variants of the wild type sequence that have no effect on the expression or function of the sheath proteins, polypeptides, or peptides or domain thereof; and mutant sequences are defined as changes in the wild-type sequence, either naturally occurring or introduced by the hand of man, that have an effect on either the expression and/or the function of the sheath proteins, polypeptides or peptides or domains thereof. The invention thus also includes the provision of DNA segments vectors, genes and coding sequence region that encode various forms of sheath proteins, polypeptides, peptides or any fusion protein, polypeptides or peptide thereof.
The sheathlin (Hu et al., 1997a) is the parent protein of sheath protein (Uchida et al., 1995) which is named as it is accumulated to the enamel sheath of immature enamel to reveal honeycomb pattern on immunohistochemical detection. The sheath protein is firstly found to be 13 kDa˜17 kDa non-amelogenin protein in newly formed enamel of pig (Fukae and Tanabe, 1987a). The sheathlin is degraded at early stage of development into three peptides, 17 kDa sheath protein, 25 kDa acidic protein and Ca binding protein (Fuake and Tanabe, 1987b).
Various therapeutic methods using the compositions of the invention contains the application for not only periodontal ligament regeneration, cementum regeneration and alveolar bone regeneration against periodontal disease and periodontitis, but also implant of artificial material like tooth and replantation of extracted tooth.
Enamel sheath protein (ESP) is involved in the construction of the enamel sheath during tooth development. The 17 kDa ESP is a one-step cleavage product processed by proteolysis from the N-terminal side of sheathlin (ameloblastin/amelin), one of the porcine enamel matrix proteins. Enamel sheath protein exhibits periodontal ligament and cementum regeneration activity in a buccal dehiscence model in dogs, and promotes the cytodifferentiation of cultured human periodontal ligament (HPDL) cells. The aim of this study was to determine the peptide segment on the C-terminal side sequence of the human ESP that possesses a cytodifferentiation activity on cultured HPDL cells.
Enamel protein extracts induce osteogenesis and/or cementogenesis. Commercially available enamel protein preparations such as Emdogain are used clinically to stimulate periodontal regeneration activity. However, the molecules and the mechanisms behind the bioactivities of these complex, multicomponent enamel protein preparations are unclear. The cementum regeneration activity, which is one 4 aspect of the periodontal ligament regeneration activity, is carried by proteins in newly formed porcine secretory stage enamel, including the 25 kDa amelogenin, 89 kDa enamelin and 17 kDa enamel sheath 5 protein (ESP).
Amelogenins are the major structural components in the secretory stage enamel matrix, in which the growth of enamel crystals occurs. Amelogenins assemble to form micelle structures, which support the elongating enamel crystal ribbons. Amelogenins are progressively degraded by the enamel proteases, Mmp-20 and Klk4 (EMSP-1). The slow degradation of amelogenin assemblies that separate the crystals provides space for the progressive thickening of the enamel crystals, which is necessary for hardening the enamel. The 89 kDa enamelin resides mainly in the enamel rod of newly formed secretory stage enamel and has high affinity for binding to the enamel crystallites. The function of enamelin is critical for proper dental enamel formation and it thus play a role in crystal formation. The 17 kDa ESP is the N-terminal cleavage product of sheathlin, which has also been called ameloblastin or amelin, and it becomes concentrated within the sheath space between enamel rods.
When these proteins are individually tested using the buccal dehiscence model in dogs, cementum regenerating activity is exhibited by 17 kDa ESP purified from newly formed secretory stage enamel, but not by the amelogenin and enamelin fractions. Significantly, 13 and 15 kDa ESPs, which are cleavage products of 17 kDa ESP lacking the C-terminal peptide, do not exhibit any cementum regeneration activity.
In cell culture systems, the alkaline phosphatase (ALP) activity is the standard marker for the cytodifferentiation of osteoblast-like cells. Various purified ESPs have been used to stimulate human periodontal ligament (HPDL) cells, which were subsequently assayed to determine their ALP activity. Only 17 kDa ESP shows cytodifferentiation activity in a cell culture system, while other lower molecular weight ESPs do not, thus suggesting that the periodontal ligament inducing activity and cytodifferentiation activity are carried by the C-terminal segment of the 17 kDa ESP that is missing from the smaller molecular weight ESPs. Synthetic peptides were synthesized that corresponded to segments of the C-terminal sequence of porcine 17 kDa ESP, and thereby showed a cytodifferentiation activity similar to the 17 kDa ESP itself when their ALP-inducing activities of HPDL cells were evaluated in a cell culture system. Peptides corresponding to this same region of human ESP were therefore synthesized to identify any peptides demonstrating cytodifferentiation activity using an HPDL cell culture system.
2. Description of Related Art
The enamel matrix proteins (enamel matrix derivatives: EMD) are used for repairing the periodontal ligament of periodontitis as one of the periodontal regeneration remedy. It is well accepted that the enamel matrix proteins have the periodontal regeneration activity (Hammarström 1997, Hammarström et al., 1997). Nevertheless, the final periodontal regeneration which gratifies patient's desires is not always accomplished. In addition there is a risk of infection of E type or unknown virus, since the extract from porcine immature enamel matrix is used for the remedy for clinical treatment, although the risk is at present avoided by heating the enamel matrix proteins before preparing the remedy. Another risk is that immunity from EMD for repeat application to the same patient is not always denied, although clinical safety of EMD is reported (Zetterstrom et al., 1997). The biggest problem is at present scarce information about periodontal regeneration promoting factor, because the enamel matrix proteins are consisted of multi components.
Enamel Matrix Proteins
There are in the enamel matrix three enamel matrix proteins and two proteolytic enzymes. The cDNA and derived amino acid sequences of these proteins and proteinases have been revealed by previous studies. In addition, two growth factors are found to be in porcine secretory enamel matrix.
The structural proteins are amelogenin, (Fukae et al., 1983; Snead et al., 1985; Shimokawa et al., 1987), enemelin (Fukae et al., 1993; Fukae et al., 1996; Hu et al., 1997b), and sheathlin (ameloblastin/amelin) (Cerny et al., 1996; Krebsbach et al., 1996; Hu et al., 1997a). In the developing enamel matrix, enamel matrix serine proteinase (EMSP) (Fukae et al., 1977; Tanabe 1983; Tanabe et al., 1996; Simmer et al., 1998) and a novel metalloproteinase (enamelysin) (Bartlett et al., 1996; Fukae et al., 1998) are cloned and characterized. It is confirmed in porcine enamel matrix the existence of osteogenetic growth factors which are bone morphogenetic factor (BMP) and transforming growth factor (TGF)-β (Suzuki et al., 2005).
Therefore, the immature enamel matrices throughout several developing stages contain a lot of amelogenin, sheathlin and enamelin derivatives. If the purification of specific protein was performed from enamel matrix protein fraction, the purification is interrupted by the causes of a lot of amelogenin gene products and their derivatives, and their aggregation nature.
Amelogenin
Amelogenins are abundant throughout the developing enamel matrix, and have an aggregation nature in solution. They aggregate to form a precipitate in neutral pH at room temperature and reversibly change their phase into the solution at lower temperature. Protein chemical analyses suggest that apparent molecular weights of 25 kDa amelogenin, one of uncleaved amelogenin polypeptides (Fukae et al., 1980; Uchida et al., 1991) is most abundant in the porcine gene products along with 27, 18 and 6.5 kDa amelogenins produced by splicing of amelogenin mRNA (Yamakoshi et al., 1994; Hu et al., 1996; Ikawa et al., 2005).
In newly formed enamel porcine 25 kDa amelogenin is converted by the cleavage of C-terminal hydrophilic domain with the action of enamelysin into 20 kDa amelogenin, and then in advanced developmental secretory enamel the 20 kDa amelogenin is splitted out into two fragments of 6 kDa and 13 kDa amelogenins by the action of the other proteinase EMSP (KLK4). The 13 kDa amelogenin is soluble in neutral solution and disappears from the system to produce the space for crystal growth during the secretory stage enamel.
Sheathlin
Sheathlin has 65 kDa molecular mass and is the parent protein of sheath protein which is named for 13˜17 kDa non-amelogenin protein found firstly in porcine newly formed enamel matrix (Fukae and Tanabe 1987a; Uchida et al., 1991). Porcine sheathlin is degraded at once after its secretion from ameloblasts into three segments, 17 kDa sheath protein derived from N-terminal side, 29 kDa calcium binding protein derived from C-terminal side (Fukae and Tanabe 1987b; Murakami et al., 1997; Yamakoshi et al., 2001) and 25 kDa acidic protein derived from middle part of molecule. The sheath protein accumulates at once to in future prism sheath space (Uchida et al., 1991; Uchida et al., 1995) and is degraded into lower molecular weight of 15 kDa and 13 kDa sheath protein in advanced developing stage of secretory enamel.
Enamelin
Enamelin is a parent protein of 155 kDa with cleavage products having apparent molecular weights of 142, 89, 56, 45, 34, 32, and 25 kDa (Fukae et al., 1996; Hu et al., 1997b). In the newly formed enamel 89 kDa enamelin is mainly existed in alkaline soluble fraction. In advanced developing secretory enamel the 89 kDa enamelin is degradated into 32 kDa enamelin which is soluble in neutral solution and has affinities for fluorohydroxyapatite (Tanabe et al., 1990) and Ca ions (Yamakoshi et al., 2001).
Growth Factors
There is additional evidence that, besides enamel proteins, potent signaling molecules may be resident in enamel extracts. Recently, bone morphogenetic protein (BMP)-like activity was deduced to be in porcine enamel extracts using ST2 cells (a mouse bone marrow stromal cell line) by the action of noggin (Iwata et al., 2002), transforming growth factor beta (TGF-β)-like activity was identified using oral epithelial and fibroblastic cells (Kawase et al., 2001). The TGF-β-like activity in the enamel matrix protein increases the ALP activity of HPDL cells, promotes their cytodifferentiation, and finally induces mineralization (Nagano 2003). The relationship between the presence of these growth factor-like activities in enamel extracts and the induction of osteogenesis and cementogenesis during periodontal regeneration is unknown.
It is shown that the periodontal ligament (PDL) is regenerated in the experimental cavities of intrabony defects created on a buccal dehiscence model in monkeys after the application of porcine enamel matrix proteins (Hammarström 1997, Hammarström et al., 1997). The idea that enamel matrix proteins are involved in the formation of cementum is based on the fact that coronal acellular extrinsic fiber cementum is formed on enamel surface in a number of species (Hammarström 1997). Application of porcine enamel matrix in experimental cavities in the roots of incisors of monkeys induces formation of acellular cementum that is well attached to the dentin. It indicates the enamel matrix proteins have the potential to induce regeneration of the same type of cementum (Hammarström 1997, Hammarström et al., 1997). The enamel matrix derivatives stimulate the proliferation and differentiation of human PDL cells (Gestrelius et al., 1997) and enhance bone formation (Boyan et al., 2000). These show that enamel proteins have bioactivities such as the induction of osteogenesis and cementogenesis.
Based on these results, the enamel proteins, enamel matrix derivatives (EMD) which is commercially available as EMDOGAIN®, are used clinically for PDL regeneration of periodontitis (Heijl 1997, Heijl et al., 1997). This new treatment induces a noteworthy result which is not obtained up to that time as the effect of periodontal disease treatment. However, when the enamel proteins are used for the treatment of periodontitis, periodontal ligament regeneration is not always accomplished to the level of expected result. It indicates that the usage of enamel proteins for the treatment of periodontal disease leaves much room for improvement.
The problem remaining to be solved is the elucidation of the real form of bioactivities contained in the enamel proteins. The researchers developed the EMD expect that the bioactivities are due to the amelogenin family a major component in enamel matrix. However, this idea is denied by finding the existence of periodontal ligament in amelogenin-deficient mice (Gibson et al., 2001). In fact, the amelogenins and their derivatives separated by ammonium sulfate precipitation fractionation (Kanazashi et al., 2004) or gel filtration system (Kanazashi et al., 2006; Fukae et al., 2006) have no cementum regeneration activity by histological analyses using experimental defects created on buccal dehiscence model of dogs.
There is another approach to estimate the bioactivities such as osteoinductive activities, BMP-like activity (Iwata et al., 2002) and TGF-β-like activity (Kawase et al., 2001). The TGF-β-like activity in the enamel matrix protein increases the alkaline phosphatase (ALP) activity of human periodontal ligament (HPDL) cells, promotes their cell differentiation and finally induces the mineralization (Nagano 2003; Nagano et al., 2004). The existences of both BMP and TGF-β in porcine enamel proteins are confirmed by lucipherase reporter assays (Suzuki et al., 2005). However, it is unclear the function of these osteoinductive factors in periodontal regeneration in in vivo system, although these may contribute to the induction of osteogenesis and cementogenesis or both and biomineralization during periodontal regeneration.
It is not doubted that the EMD has the periodontal regeneration activity. However, it is too hard to separate the activity from the EMD since the EMD consists of multi components containing a lot of amelogenins and their derivatives which have aggregation nature. To avoid the obstruction by amelogenin aggregation, the usage of newly formed secretory enamel for separating the cementum regeneration promoting factor seems to be advantageous. Because it contains the smallest amount of amelogenin degradation products comparing to the other advanced developmental immature enamel. The 0.05M carbonate buffer (pH 10.8) which inhibits the aggregation of amelogenins is employed for the separation. Over 95% of whole proteins contained in immature enamel is solubilized briefly by homogenizing in this buffer (Fukae and Tanabe, 1998).
The management to avoid the degradation of periodontal regeneration activity is employed. There are at least four proteolytic activities detected in developing dental enamel matrix. They are two metalloproteinases, gelatinase and enamelysin (MMP-20) (Bartlett et al., 1996; Fukae et al., 1998), and two serine proteinases including EMSP (KLK4) (Fukae et al., 1977; Shimizu et al., 1979; Simmer et al., 1998). Enamel matrix serine proteinase and enamelysin were cloned characterized and involved in the degradation of amelogenins and non-amelogenin proteins during not only transition stage enamel but also secretory stage enamel (Tanabe et al., 1992; Tanabe et al., 1996; Fukae and Tanabe, 1998). EMSP and proEMSP found in the secretory enamel are extracted with only the neutral phosphate buffer. The extraction of neutral soluble fraction is needed to avoid the degradation of periodontal regeneration activity, since proEMSP is activated with metalloproteinases (Tanabe et al., 1996). The action of MMP-20 found in alkaline soluble fraction was inhibited by adding at once EDTA, the inhibitor of matrix metalloproteinase, after the extraction of alkaline soluble fraction.
Complete periodontal regeneration could be accomplished by first cementum regeneration (CR) along with burying of collagenous bundles and then periodontal ligament regeneration and alveolar bone regeneration, if it is reasoned by the analogy of root formation at tooth development. Therefore, the cementum regeneration is thought to be most important part in the periodontal regeneration processes. Nevertheless, since no marker protein is found in the cementum formation, the histological analysis on buccal dehiscence model of dogs are employed for cementum regeneration (CR) activity of fractionated enamel proteins. The CR capacity of individual protein fraction obtained at each separation or purification step is evaluated step by step by the regenerated cementum of eight weeks produced on the experimental intrabony defects created along the roots of the canine mandibular premolars.
When porcine enamel proteins are separated under the difference of developmental stage and examined for their CR activity, the CR activity is found in newly formed enamel rather than the advanced developmental secretory enamel. In newly formed secretory enamel, CR activity was detected in the alkaline soluble fraction, but not in the neutral soluble fraction. When the alkaline soluble fraction was separated into 4 fractions by Sephadex G-100 gel filtration, CR activity was found in the first eluted peak (fraction 1) containing the aggregate of sheath proteins along with a small amount of amelogenins and enamelins. The other peaks consisted of amelogenins and their derivatives had no CR activity.
When the fraction 1 was separated into enamelin fraction and aggregate fraction containing sheath proteins and amelogenins, the activity was found in the aggregate fraction. It is concluded the CR activity was in sheath proteins, because the amelogenin had no CR activity. And so sheath proteins were purified in dissociative condition and homogeneous protein fraction of each 13 kDa, 15 kDa and 17 kDa sheath protein was obtained. CR activity was found only in 17 kDa sheath protein. The 17 kDa sheath protein was split into 15 kDa sheath protein by the cleavage of C-terminal side peptide and the 13 kDa sheath protein was derived from the 15 kDa sheath protein by the cleavage of N-terminal side peptide. Therefore, CR activity was existed in the sequence of C-terminal side of 17 kDa sheath protein.
After CR activity was determined to be resided in the C-terminal peptide of 17 kDa sheath protein, to search the specific sequence having CR activity was examined by the detection of alkaline phosphatase inducing activity of human periodontal ligament (HPDL) cells, after application of purified enamel proteins or their peptides on cell culture system. The employment of cell culture system is handy method to link the detection of CR promoting activity, because the increase of ALP activity of HPDL cells shown their cytodifferentiation plays important function for acellular cementum formation, deduced from morphometric evaluation in the light microscope on ALP-deficient mice (Beertsen et al., 1999). In general, it is unclear whether the evaluation of ALP activity in HPDL cell culture system contributes to the induction of osteogenesis or cementogenesis. However, CR activity resided in the 17 kDa sheath protein was determined by the regenerated cementum of eight weeks produced on the experimental intrabony defects created on the canine mandibular premolars' roots. Therefore, the increase of ALP activity of HPDL cells in the cell culture system links strongly to the evaluation of CR promoting activity of 17 kDa sheath protein or peptides. It was characterized about HPDL cells on cell culture system that ALP activity of HPDL cells induced by adding 1α-25dihydroxy-Vitamin D3 was increased by adding TGF-β, and decreased by adding BMP
The purified porcine three sheath proteins were examined their ALP inducing activity of HPDL cells in cell culture system. ALP inducing activity was found in 17 kDa sheath protein, but scarce activity was in 13 kDa and 15 kDa sheath proteins. It indicates the cytodifferentiation activity of HPDL cells is resided in the C-terminal side peptide of 17 kDa sheath protein. And so, based on the sequence of porcine or human sheath protein, the peptides were synthesized and examined their ALP inducing activity on the cell culture system. On human synthesized peptides, SEQ ID NO: 1 and SEQ ID NO: 2 were shown dose dependently to increase ALP inducing activity of HPDL cells. Since the activities of these peptides were not inhibited by TGF-β1 inhibitor, TGF-β1 receptor was not common receptor of these peptides.
As will be appreciated by persons skilled in the art, the invention also relates to protein sequences with deduced amino acid sequences of SEQ ID NO:1 to SEQ ID NO:34 which have preferably 5% or greater identity, more preferably 10% or greater identity, more preferably 15% or greater identity, more preferably 20% or greater identity, more preferably 25% or greater identity, more preferably 30% or greater identity, more preferably 35% or greater identity, more preferably 40% or greater identity, more preferably 45% or greater identity, more preferably 50% or greater identity, more preferably 55% or greater identity, more preferably 60% or greater identity, more preferably 65% or greater identity, more preferably 70% or greater identity, more preferably 75% or greater identity, more preferably 80% or greater identity, more preferably 85% or greater identity, more preferably 90% or greater identity, more preferably 95% or greater identity, more preferably 96% or greater identity, more preferably 97% or greater identity, more preferably 98% or greater identity, and more preferably 99% or greater identity.
The present invention further provides proteins that consist essentially of the amino acid sequences provided in SEQ ID NO: 1 to SEQ ID NO: 36 and SEQ ID NO: 39 to SEQ ID NO: 44. A protein consists essentially of an amino acid sequence when such an amino acid sequence is present with only a few additional amino acid residues, for example from about 1 to about 100 or so additional residues, typically from 1 to about 20 additional residues in the final protein.
The present invention further provides proteins that comprise the amino acid sequences provided in SEQ ID NO: 1 to SEQ ID NO: 36 and SEQ ID NO: 39 to SEQ ID NO: 44. A protein comprises an amino acid sequence when the amino acid sequence is at least part of the final amino acid sequence of the protein. In such a fashion, the protein can be only the peptide or have additional amino acid molecules, such as amino acid residues (contiguous encoded sequence) that are naturally associated with it or heterogeneous amino acid residues/peptide sequences. Such a protein can have a few additional amino acid residues or can comprise several hundred or more additional amino acids.
To determine the percent identity of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the length of a reference sequence is aligned for comparison purposes. The amino acid residues at corresponding amino acid positions are then compared. When a position in the first sequence is occupied by the same amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid “identity” is equivalent to amino acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
Some embodiments of the present inventions are described with reference to the numbered paragraphs below:
1. A protein comprising: an isolated sheath protein that is one of a derivative produced by the action of proteinase from a sheathlin (ameloblastin/amelin), one of structural enamel proteins; and is derived from amino-terminal side of the sheathlin.
2. The protein of paragraph 1, where the isolated sheath protein is mammalian.
3. The protein of paragraph 2, where the isolated mammalian sheath protein is porcine.
4. The protein of paragraph 2, where the isolated mammalian sheath protein is human.
5. The protein of paragraph 1, where amino acid sequence of the isolated sheath protein is selected from the group consisting of SEQ ID NO: 10 and SEQ ID NO: 11.
6. A method of producing an effect comprising administering the protein of paragraph 5 to a mammal.
7. The method of paragraph 6, where the mammal is a Human.
8. A method of producing an effect comprising administering the protein of SEQ ID NO: 11 to a mammal.
9. The method of paragraph 8, where the mammal is a Human.
10. A polypeptide or peptide segment characterized as: comprising a sequence region of at least about 3 contiguous amino acids that have the same sequence as about 3 contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13; or comprising from 3 to about 1,000 amino acids in length that synthesizes the polypeptide segment selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13.
11. The polypeptide or peptide segment of paragraph 10, wherein the segment comprises a sequence region of at least 3 contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO:13.
12. The polypeptide or peptide segment of paragraph 10, wherein the segment is from about 3 to about 1,000 amino acids in length and synthesizes an amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13.
13. The polypeptide or peptide segment of paragraph 10, wherein the segment is from about 3 to about 1,000 amino acids in length and biosynthesizes by a recombinant protein expression system to the amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 12 and SEQ ID NO: 13.
14. A method of producing an effect comprising administering the protein of paragraph 10 to a mammal.
15. The method of paragraph 14, where the mammal is a Human.
16. A polypeptide or peptide segment characterized as: comprising a sequence region of at least about 3 contiguous amino acids that have the same sequence as about 3 contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25; or comprising from 3 to about 1,000 amino acids in length that synthesizes the polypeptide segment selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25.
17. The polypeptide or peptide segment of paragraph 16, wherein the segment comprises a sequence region of at least 3 contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25.
18 The polypeptide or peptide segment of paragraph 16, wherein the segment is from about 3 to about 1,000 amino acids in length and synthesizes an amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25.
19. The polypeptide or peptide segment of paragraph 16, wherein the segment is from about 3 to about 1,000 amino acids in length and biosynthesizes by a recombinant protein expression system to the amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids of the sequence selected from the group consisting of SEQ ID NO: 24 and SEQ ID NO: 25.
20. A method of producing an effect comprising administering the protein of paragraph 16 to a mammal.
21. The method of paragraph 20, where the mammal is a Human.
22. A polypeptide or peptide segment characterized as: comprising a sequence region of at least about 3 contiguous amino acids that have the same sequence as about 3 contiguous amino acids of SEQ ID NO: 9; or comprising from 3 to about 1,000 amino acids in length that synthesizes the polypeptide segment of SEQ ID NO: 9.
23. The polypeptide or peptide segment of paragraph 22, wherein the segment comprises a sequence region of at least 3 contiguous amino acids from SEQ ID NO: 9.
24. The polypeptide or peptide segment of paragraph 22, wherein the segment is from about 3 to about 1,000 amino acids in length and synthesizes an amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids from SEQ ID NO: 9.
25. The polypeptide or peptide segment of paragraph 22, wherein the segment is from about 3 to about 1,000 amino acids in length and biosynthesizes by a recombinant protein expression system an amino acid segment of an artificial sequence comprising a sequence region of contiguous amino acids from SEQ ID NO: 9.
26. A method of producing an effect comprising administering the protein of paragraph 22 to a mammal.
27. The method of paragraph 26, where the mammal is a Human.
28. A polypeptide or peptide segment characterized as comprising a sequence region selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.
29. A method of producing an effect comprising administering the polypeptide segment of paragraph 28 to a mammal.
30. The method of paragraph 28, where the mammal is a Human.
31. A composition comprising an isolated aggregate that comprises a sheath protein.
32. The composition of paragraph 31, where the isolated aggregate forms with sheath proteins and amelogenins in an alkaline solution.
33. The composition of paragraph 31, where the isolated aggregate resides in outer layer enamel, newly formed enamel, corresponding to approximately 30 μm thickness from the surface of secretory stage enamel of mammal.
34. The composition of paragraph 31, where the isolated aggregate is prepared from the outer layer enamel, newly formed enamel.
35. The composition of paragraph 31, where the isolated aggregate is separated using a method selected from the group consisting of gel filtration in alkaline solution, ion exchange chromatography, and ammonium sulfate fractionation.
36. A method of producing an effect comprising administering the protein of paragraph 31 to a mammal.
37. The method of paragraph 36, where the mammal is a Human.
38. A composition comprising isolated enamel proteins that comprise a sheath protein.
39. The composition of paragraph 38, where the isolated enamel proteins are prepared from the outer layer enamel, newly formed enamel, corresponding to approximately 30 μm thickness from the surface of secretory stage enamel of mammal.
40. The composition of paragraph 38, where the isolated enamel proteins are extracted by an alkaline solution after the extraction of neutral soluble proteins from the outer layer enamel, newly formed enamel.
41. A method of producing an effect comprising administering the protein of paragraph 38 to a mammal.
42. The method of paragraph 41, where the mammal is a Human.
This invention is not limited to specific polypeptides or peptides and the numerous modifications and variations therein will be apparent to those skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.